This proposal seeks to overcome the limitations of current and emerging multiplexed technologies that are critical to improving cancer diagnosis and treatment. These technologies utilize classical antibodies for biomarker detection, and as such are hampered by scarcity of species in which the antibodies are raised, steric hindrance, harsh multiplexing conditions, and permanent modification of the cell or tissue sample. To overcome these limitations and simultaneously enable multiplex analysis of living cells, GE will develop labile antibodies with controllable affinity. Specifically, for demonstration purposes, existing antibodies against HER2 will be modified and the resulting conjugates will be prepared at varying degrees of modification and characterized by UV-vis spectroscopy, LDS-PAGE and MALDI-TOF-MS. These conjugates will then be evaluated for their capability to bind HER2 using fixed SKOV-3 cell pellets by fluorescence microscopy and the binding kinetics will be quantified via surface plasmon resonance (Biacore). The resulting information will be used to prepare improved anti- body conjugates for validation of the technology's multiplexing capabilities. Specifically, sequential detection of HER2 and Ki67 biomarkers will be performed in clinically-relevant human breast cancer samples using primary antibodies from a single species. The specific aims of the work will be to prepare and characterize a library of modified anti-HER2 antibodies, identify and optimize lead antibody conjugates, and to validate the multiplexing capabilities of the lead antibody conjugates on clinically-relevant tissue samples. Successful development of the proposed technology will allow unlimited multiplexing with the primary antibodies from the same species;detection and quantification of multiple closely-spaced biomarkers in the same tissue sample;assessment of biomarker spatial information;correlation of biomarkers to each other, to disease progression, and to treatment response;and the opportunity to perform multiplex analysis on living cells in addition to fixed tissue. PUBLIC HEALTH RELEVANCE: The ability to screen tissue samples or individual fixed or living cells for biomarkers in a multiplexed fashion is critical for enhanced cancer diagnostics and the development of more targeted therapies. The proposed project will achieve this by chemically modifying antibodies targeted against disease biomarkers to achieve controllable affinity, which will enable efficient binding and release. These antibodies will preserve the integrity of the tissue or cell sample while enabling potentially unlimited multiplexing capabilities without restrictions due to limited antibody-species availability or overlapping biomarker epitopes.